FLT3 is the most frequently mutated gene in cases of acute myelogenous leukemia (AML). About 30-35% of patients have either internal tandem duplications (FLT3-ITD) in the juxtamembrane (JM) domain or mutations in the activating loop of FLT3, the consequences of which are constitutive activation of the tyrosine kinase. FLT3 activating mutations have also been described in acute lymphoblastic leukemias including subsets harboring 11q23 rearrangements implicating it in the pathogenesis of a broader spectrum of hematopoietic neoplasms. Significantly these mutations confer a poor clinical prognosis to patients in most retrospective studies providing a compelling basis for understanding the pathogenesis of FLT3-ITD mediated disease and developing and testing new therapies that can specifically target the FLT3 molecule. The research described in this 5-year proposal outlines specific aims designed to investigate the in vivo effects of an activating FLT3-ITD mutation in a whole animal system through the characterization of a novel FLT3-ITD knock-in mutant mouse model which we have generated using a combination of standard transgenic and embryonic (ES) cell gene-targeting techniques. Expression of activated FLT3 from the endogenous murine FLT3 promoter in these animals provides distinct advantages over retroviral transduction models where differences in expression levels of activated FLT3 may affect disease phenotype and avoids potential mutations introduced by retroviral integration. These animal models should provide insights not only into how activated FLT3 contributes to the pathogenesis of leukemia, but also into the biological role of this gene in normal hematopoiesis. Moreover, these mice should provide powerful reagents that can be used to assess the efficacy of targeted drug therapy against FLT3-induced leukemias including the use of both currently established FLT3 inhibitors and new generation high affinity FLT3 inhibitors. Finally, these mice can be employed with other established mouse model systems to further our understanding of how different classes of mutations can cooperate with one another in the development of acute leukemia. Specific aims include: (1) To characterize a FLT3-ITD knock-in mouse model including (a) assessing effects of FLT3-ITD expression from the endogenous FLT3 promoter on hematopoietic progenitor development and (b) detailed characterization of mono-allelic and bi-allelic FLT3-ITD knock-in phenotypes; (2) To assess the therapeutic efficacy of small molecule FLT3 tyrosine kinase inhibitors in animal drug trials with FLT3-ITD mouse lines; and (3) To investigate a cooperative model of acute leukemia by crossing FLT3-ITD mice with other leukemogenic mouse models including PML/RARalpha, AML1/ETO, and C/EBPalpha p30 transgenic mouse lines and assessing for therapeutic efficacy with novel small molecule inhibitors.